Use of Emu Oil and its various fractions as a carrier for antifungal, antibacterial, and antiviral medications and preparations

ABSTRACT

An animal-derived lipid is disclosed that is useful as a carrying agent for anti-microbial formulations. Pharmaceutical and other preparations including Emu Oil are also described as profoundly useful components in anti-bacterial, anti-fungal, and anti-viral treatments. This lipid material is extracted from the Emu ( Dromais Novae - Hollandiae ), an indigenous bird of Australia and New Zealand. Therapeutic compositions comprising Emu Oil in combination with an extracellular product of  Bacillus coagulans  or  Pseudomonas lindbergii  strain, comprising a supernatant or filtrate of said culture suitable for topical application to the skin or mucosal membranes of a mammal are utilized to inhibit the growth of bacterium, yeast, fungi, virus, and combinations thereof. Additionally, the aforementioned therapeutic composition may also include an anti-microbial, anti-mycotic, and/or anti-viral agent. Methods of treatment and therapeutic systems inhibit the growth of bacterium, yeast, fungi, virus, and combinations thereof, by topical application of therapeutic compositions comprising Emu Oil in combination with an extracellular product of  Bacillus coagulans  or  Pseudomonas lindbergii  strain suitable for topical application to the skin or mucosal membranes of a mammal. Similarly, the aforementioned method may also employ a therapeutic composition additionally containing an anti-microbial, anti-mycotic, and/or anti-viral agent.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/843,277 filed May 11, 2004 now U.S. Pat. No. 7,048,950, which is acontinuation application of U.S. Ser. No. 10/384,840 filed Mar. 10,2003, now U.S. Pat. No. 6,733,751, which is a divisional application ofU.S. Ser. No. 09/850,466, filed May 7, 2001, now U.S. Pat. No.6,531,126, which is a continuation application of U.S. Ser. No.09/384,043, filed Aug. 26, 1999, now abandoned, each of which areincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods of use for thetreatment of bacterial, fungal, and viral infections of the dermis andcuticle. More specifically, the present invention relates tocompositions and methods of use of Emu Oil, and it various associatedfractions, in combination with the appropriate medicaments, in thetreatment of bacterial, fungal, and viral infections of the dermis andcuticle. The present invention also relates to the utilization oftherapeutic compositions comprised of Emu Oil in combination with aprobiotic, viable Bacillus bacteria, spores, and extracellularsupernatant products, as well as the extracellular product ofPseudomonas lindbergii, as a topical agent for the prevention and/orcontrol of infections caused by bacterium, fungi, yeast, and virus, andcombinations thereof.

BACKGROUND OF THE INVENTION

1. Emu Oil

Emu Oil, is an animal-derived lipid composition, extracted from the Emu(Dromais Novae-Hollandiae), a flightless bird part of a group calledratites (which also includes the ostrich and the kiwi), indigenous toAustralia and New Zealand.

Emu Oil is extracted from a thick fat-pad on the back of the bird whichputatively functions to protect the animal from the extreme temperaturesin its Australian homeland. The fat is carefully extracted to preventthe formation of trans-fatty acids, wherein approximately 100 pounds offat produces approximately 50 to 90 pounds of unrefined, pale yellowoil. The chemical composition and characteristics of Emu Oil has beenquantitatively ascertained and is set forth below in Table I.

TABLE I Fatty Acid Composition: C-14:0 (Myristic):  0.4% C-16:0(Palmitic): 21.5% C-16:1 (Palmitoleic):  3.7% C-18:0 (Stearic): 10.6%C-18:1 (Oleic): 51.4% C-18:2 (Linoleic): 12.7% C-18:3 (Linolenic):  0.9%Calculated Iodine Value: 69.7 Free Fatty Acid: 0.33% Acid Value: 0.66%Peroxide Value: 1.53% Moisture: 0.03% Refractive Index @ 40° C.:1.4606% 

As illustrated in Table I, when correctly extracted and processed, EmuOil is comprised of approximately 50% to 70% monounsaturated fattyacids, with the rest being both saturated and polyunsaturated fattyacids (see e.g., American Emu Association News, March 1995). Emu Oil isalmost purely triglyceride in nature, which makes it an almostcompletely neutral lipid. In addition, the monounsaturated fatty acid,oleic acid, is the largest single fatty acid component of Emu Oil.Traditional beliefs of geographically widely-separated AustralianAboriginal communities agree on the beneficial properties of Emu Oil asa natural remedy. The oral history of the Australian Aboriginesindicates their use of Emu Oil for over forty thousand years to reducepain and stiffness in sore muscles and joints, to help expedite woundhealing, as a dermal protectorate from the effects of wind and sun, andin the treatment of bruised subcutaneous tissue, burns and dry skinproblems. Methods of administration are quire varied. For example,Aborigines have revealed methods of treatment which included hanging anEmu skin on a tree to collect the oil, and wrapping the affected area onthe individual in a freshly-killed Emu skin. However, it is believedthat in both of the aforementioned scenarios, the catalyst of the suns'heat was used to liquefy the Emu fat and enhance its absorptionqualities.

Documented records of the utilization of Emu Oil may be antedated wellover 100 years (see e.g., Whitehouse, et al., 1996. Concerning Emu Oiland its anti-arthritic activity. Fifth Queensland Poultry ScienceSymposium, Gatton College). The use of Emu Oil was among many naturalremedies adopted by settlers from the original inhabitants of Australia.The first report known was published in the Australian Post regardingexperiments by Dr. Peter Gosh (Raymond Purves Bone and Joint ResearchLaboratories, University of Sydney at the Royal North Shore Hospital)and Dr. Michael Whitehouse (Department of Pathology, University ofAdelaide), wherein the Emu Oil was required to be massaged vigorouslyonto the sore muscle or joint and the process repeated as often asrequired, hence pressure, heat and duration of rubbing were all deemedto be relevant factors.

This, although Emu Oil has been previously described, the majority ofits uses or properties/characteristics is anecdotal in nature. Theseuses and properties include (see e.g., DuBois, 1999. Explore Issue8:1-10): (i) its ability to act as a dermal penetrant and medicamentcarrier; (ii) its anti-inflammatory properties; (iii) its ability to actas an emollient/emulsifier; (iv) its bacteriostatic properties; (v) itslow potential for irritation of the skin; (vi) its non-comedogenicproperties (i.e., it does not clog up pores); and (vii) itsmoisturizing, wound-healing, general “anti-aging” properties. However,the quantitative information currently available almost exclusivelyrelates to the benefits of Emu Oil as an anti-inflammatory agent forarthritis, its uses for cardiovascular health when ingested, which issimilar to the use of Omega-3 fish oils to improve high-densitylipoprotein (HDL) cholesterol, and its moisturizing and general“anti-aging” properties.

There is much anecdotal material available on the anti-inflammatoryabilities of Emu Oil. It has been shown to reduce pain, swelling andstiffness in joints, to reduce recent bruising and muscle pain, and easesports related muscle strains as well. Studies have shown that differentEmu Oils (i.e., oils which were extracted by different methodologies,from different sources, and the like) possessed different levels ofanti-inflammatory ability. The ability of Emu Oil to penetrate thestratum corneum dermal barrier and concomitantly act as a carrier, makesit highly valuable for use in therapeutic compounds in the preventionand/or treatment of a variety of conditions. This ability is believed tobe primarily due to both its extremely high content of oleic acid and atotal lack of indigenous phospholipids. Accordingly, Emu Oil could becombined with various medicinals or cosmetic materials to facilitatetheir ability to penetrate this layer of keritinized tissue in a moreefficacious and cost-effective manner than the currently-utilizedliposome- and iontophorisis-based technologies. For example, the abilityof Emu Oil to act as a trans-dermal penetrant with respect toKetoprofen, a well known non-steroidal, anti-inflammatory drug (NSAID)found in Actron™ and like products, was examined in a recent studyperformed at Auburn University. Ketoprofen is one of the proprionic acidderivative drugs, which have been utilized in numerous Europeancountries for more than 15 years as an effective treatment forrheumatoid arthritis and osteoarthritis. Although it is available inmore than 80 countries throughout the world, it did not receive approvalfor over-the-counter (OTC) use in the United States until 1996. AlthoughKetoprofen is readily absorbed, it frequently produces a number ofadverse side-effects in the gastrointestinal tract when taken orally.Moreover, the oral administration of Ketoprofen has also been associatedwith such serious deleterious physiological side-effects as renaldysfunction, marked edema, and hepatic dysfunction (e.g., jaundice). Theutilization of a topically-administered Ketoprofen composition to thedermis over the inflamed tissues or joints would perhaps mitigate someof the aforementioned side-effects and may also potentially result inthe accumulation of the drug within associated synovial tissues, thesite of the desired anti-inflammatory reaction. However, recent studiesin which Ketoprofen was topically-administered without the utilizationof dermal-penetrants (e.g., Emu Oil) demonstrated that this compound wasadsorbed through viable, keritinized dermal tissue in a very limitedconcentration, if at all.

Conversely, the results demonstrated that the concomitant utilization ofa dermal-penetrant produced markedly elevated adsorption of thecompound. Specifically, an Emu Oil—propanol—Ketoprofen combination wasshown to produce a 3-times higher serum levels in mice followingtrans-dermal application, than either a DMSO—Bovine Serum—Ketoprofen oran Isopropyl alcohol—Ketoprofen combination. This result wasparticularly encouraging due to the fact that Emu Oil was approved bythe FDA for human use in July of 1992, and DMSO has not yet receivedsuch approval.

In a related study, the ability of Emu Oil to decrease the concentrationof inflammatory molecules was examined (see Smith and Craig-Schmidt, AEAConvention Las Vegas, Nev. (Jun. 6-8, 1995)). Eicosanoids arehormone-like compounds synthesized from essential fatty acids and havebeen demonstrated to be synthesized in dermal tissue (see e.g.,Wilkerson and Walsh, 1977. J. Invest. Dermatol. 68: 210-214). While someof these compounds serve normal physiological functions, others areinvolved in the inflammatory response. In this study, prostaglandin F2a(PGF2a.) was utilized as an indicator of ecosanoid synthesis within thedermal tissue. The topical administration of was shown to decreaseecosanoid production in skin, as reflected by suppression of PGF2a. Thisresult may offer a possible biochemical explanation for the reportedbeneficial effects of topically administered Emu Oil.

Additionally, in 1995, Australian researchers isolated a component inEmu Oil which appears to be at least one of the active ingredientsdirectly responsible the oil's anti-inflammatory activity. Thus, thissubstance could potentially be utilized to develop or isolate additionalanti inflammatory medications which are without deleteriousphysiological side-effects, are non-irritating, which possess long-termbiological and physiological activity, and which are far less expensivethan currently-utilized anti-inflammatory regimens.

Emu Oil also possesses a high degree of emollient/emulsificationproperties, and hence has good “blendability”. In practice this meansthat Emu Oil has the ability to blend or make oil and water misable,producing a cream that does not feel oily on the skin. One inherentproblem is that most creams do not penetrate the dermal barrier, howeverthis is ameliorated by the utilization of Emu Oil without leaving anoily residue behind. This bodes very well for its future use in both thecosmetic and pharmaceutical industries.

An additional property of Emu Oil is that it is bacteriostatic. Recentstudies have demonstrated that in its pure state, Emu Oil grows nobacterial organisms. Thus, pure non-contaminated Emu Oil has a longshelf-life due to its bacteriostatic nature and due to its low levels ofpolyunsaturated fats which are the most subject to oxidation andeventual rancidity. Similarly, Emu Oil's bacteriostatic activity will beof useful in both cosmetic and pharmaceutical industries.

Emu Oil also possesses an extremely low potential for irritation of theskin. Moreover, it has also been shown to have almost no side-effects,which means that (even at full strength), Emu Oil has irritation levelsso low that they are the same as those found in putting water on theskin (i.e., is practically nonexistent). This characteristic is unusual,as most anti-inflammatory drugs are irritating, when applied topically,and possess side-effects.

Emu Oil is non-comedogenic in nature, and does not “clog” the pores ofthe skin nor tend to cause acne when used. This tendency cannot be saidfor, e.g., mineral oil (which is one of the current, popular carrieroils in cosmetics and rubbing oils) which frequently causes outbreaks ofacne when used.

Finally, Emu Oil is a highly efficacious moisturizing agent, which addsto its protective ability and promotes anti-aging of the skin.Researchers believe that its unique combination of saturated andunsaturated fatty acids may be an explanation for its ability to enhancethe willingness of the upper layers of the skin to retain water. Forexample, application of Emu Oil has been demonstrated to increase theoverall thickness of human skin by approximately 2.5-times, thusreducing its tendency to form “wrinkles”. In addition, there is muchanecdotal information regarding the anti-aging and wound healingabilities of Emu Oil. A double-blind study is currently being performedat the Timothy J. Harner Burn Center (affiliated with the UniversityMedical Center in Lubbock, Tex.) to authenticate this anecdotalmaterial.

The general “anti-aging” properties of Emu Oil was examined at theBoston University School of Medicine. In this double-blind study, arefined Emu Oil known as Kalaya (New World Technology; Los Angeles,Calif.) was topically-administered daily to depilated mice, over atwo-week time-period. Corn oil was utilized as the negative controlsubstance. Results demonstrated that the refined Emu Oil produced a 20%increase in the overall rate of DNA synthesis within the skin cells ofthese animals, whereas the rate of DNA synthesis within the negativecontrol animals remained normal. A marked increase in the overallthickness of the skin, to which the Emu Oil had been applied, was alsofound. In addition, over 80% of hair follicles which were quiescent atthe time of the initiation of the study, were stimulated by theapplication of the Emu Oil and began to produce a viable hair shaft.Typically, hair follicles go through stages from a quiescent phase, toan active hair-growth phase, and back to the quiescent phase again. Theadministration of Emu Oil was found to not only stimulate the hairfollicles into the active phase, but it kept them in this phase duringthe entire period of administration, as well.

Studies regarding the properties of Emu Oil have expanded to prominentnoted facilities/groups including, but not limited to: AuburnUniversity; The Arthritis Clinic, Ardmore, Okla.; Texas TechnicalUniversity; Timothy J. Harnar Burn Center; and Iowa State University.

The use of Emu Oil in veterinary medicine has also gained favor (seee.g., Zimmer, 1999. J. Equine Med. 56: 112-117). Emu Oil is frequentlyused in combination with DMSO or dexamethasone, or Gentamicin for themanagement of wounds. The treatment of non-suturable wounds (e.g.,distal leg wounds where there is decreased muscle mass), by twice-dailyapplication of Emu Oil was shown to markedly increase epithiliazation ofthese wounds, while concomitantly reducing the development of necrotictissue and scarring. Similarly, the frequency of dehiscence of suturedwounds was also demonstrated to be markedly reduced in Emu Oil-treatedequines. Emu Oil in combination with NSAID is also used to controlstiffness and pain in those affected joints in lame or arthritic horses.A frequent winter lesion seen in dairy cattle is frosted teat ends,wherein the teat end freezes and skin around the teat sloughs. Topicaladministration of Emu Oil has been found to accelerate the healingprocess and allows the continued milking of the cow during this process.The bacteriostatic properties of Emu Oil is also effective in theprevention and/or treatment of infections of the teat in dairy cows dueto milk residues. Similarly, Emu Oil is more effective in the treatmentof ringworm lesions (commonly seen in calves) than other conventionaltechniques (e.g., bleach, iodine preparations, and the like). Anotherarea in which Emu Oil is utilized in veterinary medicine is thetreatment of lesions or sores caused by casts. When a cast area isapplied it frequently retains moisture or causes pressure on bonyprotuberances, resulting in the formation of dermatitis or cast sores.Following the removal of the cast, the use of Emu Oil greatlyaccelerates the healing process of these aforementioned sores.

2. Dermal Infections

Dermal infections, especially those caused by mycotic pathogens, make-upa considerable percentage of the sale of prescription andover-the-counter medications that are sold annually worldwide. Accordingto the Center for Disease Control and Prevention (CDCP), there iscurrently a dramatic rise in the number of reported mycotic andbacterial skin infections. Annual sales of dermal and cuticularanti-fungal agents is currently exceeding two billion U.S. dollars eachyear. Moreover, dermal mycotic illness was recently shown to beincreasing at a rate of approximately 9% to 15% per annum, dependingupon the specific pathogen and disease. One of the primary factorsresponsible for the growth of these markets is the fact that more fungalpathogens are becoming resistant to the commonly-utilized anti-fungalagents each year. Examples of anti-fungal agents which arecommonly-utilized, include, but are not limited to: Fluconazole(Diflucan®; Pfizer Pharmaceutical), Intraconazole (Sporonox®; JanssenPharmaceutical), Miconazole Nitrate, Ketoconazole, Tolnaftate, Lamasil,Griseofulvin, Amphotercin B, and other compounds and the formulationsthereof.

New generations of anti-fungal and anti-bacterial drugs and preparationsare being developed every year to replace those medication in whichpathogens have become resistant. As the search for more effectiveanti-microbial agents continues, so does the search for “carryingagents” which are utilized to disperse and facilitate penetration ofthese medications through the various dermal and cuticular membranes andtissues. However, to date there has been little success in finding anagent that is able to penetrate dense cuticular material such asfinger/toenails and animal hooves.

Diseases that are most common to human dermal and cuticular membranesinclude: (i) Candidaiasis (e.g., caused by Candida albicans, Candidatropicalis, Candida golbratta, Candida parapsilosis); (ii) Tinealdiseases, also known as Athletes Foot (Tinea Pedis), Jock Itch (TineaCruis), Scalp Infection (Tinea Capitis), Ring Worm, and Beard infections(Tinea Barbae), are all caused by the Trichophyton species, including,but not limited to: Trichophyton mentagrophytes; (iii) diseases whichare caused by bacterial pathogens, including, but not limited to:Pseudomonas aeruginosa, Staphylococcus aerues, Staphylococcusepidermidus, and Propionibacterium acnes; and (iv) diseases which arecaused by viral pathogens, including, but not limited to: Herpes simplexI & II, and Herpes zoster. Perhaps one of the most difficult-to-treatdiseases of fungal etiology are fungal infections of the toenail orfingernail (i.e., Onychomycosis) due to the inability of thecurrently-available therapeutic compositions to penetrate the dermis orcuticle. The pathogen most commonly associated with this very difficultto treat disease is Trichophyton rubrum.

In animals, the most common dermal fungal disease is Ring Worm. Inanimal hooves, especially athletic equine, there are several diseases ofthe hoof that are potentially quite serious and difficult to treat,including: White Line Disease (also known as “Seedy Toe”), Hoof Thrush(another yeast- or Candida-related malady), and Drop Sole. In addition,Clubbed Foot is another dermal fungal disease that is of significantconcern to the equine industry.

3. Bacillus coagulans

Bacillus coagulans is a non-pathogenic gram positive spore-formingbacteria that produces L(+) lactic acid (dextrorotatory) inhomofermentation. This microorganism has been isolated from naturalsources, such as heat-treated soil samples inoculated into nutrientmedium (see e.g., Bergey's Manual of Systemic Bacteriology, Vol. 2,Sneath, P. H. A., et al., eds., (Williams & Wilkins, Baltimore, Md.,1986)). Purified Bacillus coagulans strains have served as a source ofvarious enzymes including, but not limited to: restriction endonucleases(see U.S. Pat. No. 5,200,336); amylase (see U.S. Pat. No. 4,980,180);lactase (see U.S. Pat. No. 4,323,651); and cyclo-malto-dextringlucano-transferase (see U.S. Pat. No. 5,102,800). Bacillus coagulanshas been used to produce lactic acid (see U.S. Pat. No. 5,079,164). Inaddition, a strain of Bacillus coagulans (designated Lactobacillussporogenes, Sakaguti & Nakayama (ATCC 31284)) has been combined withother lactic acid-producing bacteria and Bacillus natto to produce afermented food product from steamed soybeans (see U.S. Pat. No.4,110,477). Bacillus coagulans strains have also been used as animalfeed additives for poultry and livestock to reduce disease and improvefeed utilization and to, therefore, increase growth rate in the animals(see PCT Patent Application Nos. WO 9314187 and WO 9411492).

DESCRIPTION OF THE FIGURES

FIG. 1: illustrates various metabolic activities and the associated,characteristic physiological or biochemical response in Bacilluscoagulans.

FIG. 2: illustrates the various pathogens, which may be treated by useof the therapeutic compositions of the present invention, and theirassociated disorders.

FIG. 3: illustrates, in tabular form, a comparison of the anti-mycotic,Fluconazole with Bacillus coagulans and Pseudomonas lindbergiisupernatants (generically designated Ganeden Supernatant) in theinhibition of various bacterial, fungal, and yeast species.

FIG. 4: illustrates the tested fungal strains of Trichophyton species,their ATCC accession numbers, and the results of in vitro inhibition byBacillus coagulans.

FIG. 5: illustrates the tested yeast pathogen strains, their ATCCaccession numbers, and the results of in vitro inhibition by Bacilluscoagulans.

SUMMARY OF THE INVENTION

The present invention discloses the discovery that numerousdermally-associated animal diseases can be mitigated and/or preventedwhile concomitantly maintaining dermal and cuticular health by use of acombination of active agents within a therapeutic composition whichincludes: anti-fungal, anti-bacterial, or anti-viral agents comprisingorganic molecules, proteins and carbohydrates and/or bacterialfermentation products in combination with Emu Oil and its variousassociated fractions. These therapeutic composition comprise thefermentation products of specific bacterial strains in combination withan effective amount of Emu Oil in a pharmaceutically-acceptable catersuitable for administration to the dermal and/or cuticular membranes ofan animal.

In another embodiment of the present, the active anti-microbial agent isa quatenary ammonium chloride. In another embodiment, the activeanti-microbial agent is an Iodine or iodifer compound such as Betadine™.In another embodiment, the active anti-microbial agent is a phenoliccompound. In another embodiment, the active anti-microbial agent is aethanol, isopropyl or other alcohol compound or tincture. In anotherembodiment, the active anti-microbial agent is a systemic anti-fungalcompound such as Amphotericin B, Dapsone, Fluconazole, Flucytosine,Griseofulvin, Itraconazole, Kietoconazole, Miconazole, KI. In anotherembodiment, the active anti-microbial agent is a topical anti-fungalcompound such as Amphotericin B, Carbol-Fuchsin, Ciclopirox,Clotrimzole, Econazole, Haloprogin, Ketoconazole, Mafenide, Miconazole,Naftifine, Nystatin, Oxiconazole, Silver Sulfadiazine, Sulconazole,Terbinafine, Tioconazole, Tolnafiate, Undecylenic acid. In anotherembodiment, the active anti-microbial agent is a anti-fungal vaginalcompound such as Butoconazle, Clotrimazole, Econazole, Gentian Violet,Miconazole, Nystatin, Terconazole, Tioconazole.

In a preferred embodiment of the present invention, a therapeuticcomposition comprising an extracellular product of Bacillus coagulans orPseudomonas lindbergii species in a pharmaceutically-acceptable carriersuitable for topical application to skin or a mucosal membrane of amammal and Emu Oil for use in the prevention and/or control ofinfections caused by bacterium, fungi, yeast, and virus, andcombinations thereof, is disclosed. In this preferred embodiment, theextracellular product comprises the supernatant or filtrate of a cultureof a Bacillus coagulans or Pseudomonas lindbergii species.

In further embodiments of the present invention, methods for inhibitinggrowth of bacteria, yeast, fungus, virus or a combination thereof, areprovided, and include the steps of applying topically to skin or amucous membrane a composition comprising an extracellular product of aBacillus coagulans or Pseudomonas lindbergii strain, and allowing thecomposition to be present for sufficient time to inhibit growth ofbacteria, yeast, fungus, virus or any combination thereof. In oneembodiment, the applying step includes applying the composition in theform of a cream, lotion, gel, oil, ointment, suspension, aerosol spray,powder, aerosol powder or semi-solid formulation.

According to yet another aspect of the invention, there is provided acomposition comprising an extracellular product of a Bacillus coagulansor Pseudomonas lindbergii is applied to a flexible article that isintended to be worn by or attached to skin or a mucous membrane of amammal to allow probiotic activity of the bacteria to occur adjacent toor on the skin or mucous membrane.

In another embodiment of the invention, there is provided a method ofinhibiting growth of bacteria, yeast, fungus, virus or any combinationthereof, including the steps of applying a composition comprising aBacillus species or the extracellular product of a Bacillus coagulans orPseudomonas lindbergii to a solid surface, contacting the solid surfaceto skin or a mucous membrane of a mammal, and allowing the solid surfaceto contact the skin or mucous membrane for sufficient time to allowinitiation of probiotic activity of the isolated bacteria oranti-microbial properties of the extracellular product to inhibit growthof bacteria, yeast, fungus, virus or a combination thereof adjacent toor on the skin or mucous membrane. In one embodiment, the applying stepincludes applying the composition to a diaper, pliable material forwiping skin or a mucous membrane, dermal patch, adhesive tape, absorbentpad, tampon or article of clothing. In another embodiment, the applyingstep includes impregnating the composition into a fibrous or non-fibroussolid matrix.

The present invention also discloses a therapeutic system for treating,reducing or controlling microbial infections comprising a containercomprising a label and a therapeutic composition as described herein,wherein said label comprises instructions for use of the composition fortreating infection.

It should be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Unless expressly stated otherwise,the techniques employed or contemplated herein are standardmethodologies well known to one of ordinary skill in the art. Theexamples of embodiments are for illustration purposes only. All patentsand publications cited in this specification are incorporated byreference.

As utilized herein, the term “probiotic” refers to microorganisms (e.g.,bacteria, yeast, viruses, and/or fungi) which form, at a minimum, a partof the transient or endogenous flora and, thus, possess a beneficialprophylactic and/or therapeutic effect upon the host organism.Probiotics are generally known to be clinically-safe (i.e.,non-pathogenic) by those skilled within the art. Although not wishing tobe bound by any particular mechanism, the probiotic activity of Bacillusspecies is thought to result from competitive inhibition of growth ofpathogens due to superior colonization, parasitism of undesirablemicroorganisms, lactic acid production and/or other extracellularproducts having anti-microbial activity, or combinations thereof.

As utilized herein, the term “microbial” refers to bacteria, yeast,fungi, and/or virus.

The present invention discloses therapeutic compositions, methods ofuse, and articles of manufacture of a therapeutic composition comprisingan extracellular product of Bacillus coagulans or Pseudomonas lindbergiispecies in a pharmaceutically-acceptable carrier suitable for topicalapplication to skin or a mucosal membrane of a mammal and Emu Oil foruse in the prevention and/or control of infections caused by bacterium,fungi, yeast, and virus, and combinations thereof, is disclosed. In thispreferred embodiment, the extracellular product comprises thesupernatant or filtrate of a culture of a Bacillus coagulans orPseudomonas lindbergii species.

1. Lactic Acid-Producing Bacterial Strains

Typical lactic acid-producing bacteria useful as a probiotic of thisinvention are efficient lactic acid producers which includenon-pathogenic members of the Bacillus genus which produce bacteriocinsor other compounds which inhibit the growth of pathogenic organisms.Exemplary lactic acid-producing, non-pathogenic Bacillus speciesinclude, but are not limited to: Bacillus coagulans; Bacillus coagulansHammer; and Bacillus brevis subspecies coagulans.

Exemplary lactic acid-producing Lactobacillus species include, but arenot limited to: Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus DDS-1, Lactobacillus GG, Lactobacillus rhamnosus,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus gasserii,Lactobacillus jensenii, Lactobacillus delbruekii, Lactobacillus,bulgaricus, Lactobacillus salivarius and Lactobacillus sporogenes (alsodesignated as Bacillus coagulans).

Exemplary lactic acid-producing Sporolactobacillus species include allSporolactobacillus species, for example, Sporolactobacillus P44.

Exemplary lactic acid-producing Bifidiobacterium species include, butare not limited to: Bifidiobacterium adolescentis, Bifidiobacteriumanimalis, Bifidiobacterium bifidum, Bifidiobacterium bifidus,Bifidiobacterium breve, Bifidiobacterium infantis, Bifidiobacteriuminfantus, Bifidiobacterium longum, and any genetic variants thereof.

Several Bacillus species which are preferred in the practice of thepresent invention, include, but are not limited to the lacticacid-producing Bacillus coagulans and Bacillus laevolacticus.Additionally, probiotic Bacillus coagulans is non-pathogenic and isgenerally regarded as safe (i.e., GRAS classification) by the U.S.Federal Drug Administration (FDA) and the U.S. Department of Agriculture(USDA), and by those individuals skilled within the art. Various othernon-lactic acid-producing Bacillus species may be utilized in thepresent invention so long as they produce compounds which possess theability to inhibit pathogenic bacterial or mycotic growth. Examples ofsuch suitable non-lactic acid-producing Bacillus include, but are notlimited to: Bacillus subtilis, Bacillus uniflagellatus, Bacilluslateropsorus, Bacillus laterosporus BOD, Bacillus megaterium, Bacilluspolymyxa, Bacillus licheniformis, Bacillus pumilus, and Bacillussterothermophilus. Other strains that could be employed due to probioticactivity include members of the Streptococcus (Enterococcus) genus. Forexample, Enterococcus faecium, is commonly used as a livestock probioticand, thus, could be utilized as a co-administration agent. It should benoted that, although exemplary of the present invention, Bacilluscoagulans is only utilized herein as a model for various otheracid-producing (e.g., lactic acid) species of lactic acid-producingbacteria which may be useful in the practice of the present invention,and therefore is not to be considered as limiting.

The growth of these various Bacillus species is generally well-knownwithin the art. It should be noted that the exemplary culture andpreparative methods which are described herein for Bacillus coagulansmay be readily utilized and/or modified for growth and preparation ofthe other lactic acid-producing bacteria disclosed in the presentinvention.

2. Bacillus coagulans

Although, as disclosed herein, any of the aforementioned Bacillusstrains may be utilized in the practice of the present invention,purified Bacillus coagulans is exemplary and preferred as a probioticfor biological control of various microbial pathogens. Because Bacilluscoagulans forms heat-resistant spores, it is particularly useful formaking pharmaceutical compositions for treating microbial infections.Topical formulations which include viable Bacillus coagulans spores in apharmaceutically-acceptable carrier, are particularly preferred formaking and using preventive and therapeutic compositions of the presentinvention. The term “topical” is broadly utilized herein to include bothepidermal and/or skin surfaces, as well as mucosal surfaces of the body.

2.1 Characteristics and Sources of Bacillus coagulans

The Gram positive rods of Bacillus coagulans have a cell diameter ofgreater than 1.0 μm with variable swelling of the sporangium, withoutparasporal crystal production. Bacillus coagulans is a non-pathogenic,Gram positive, spore-forming bacteria that produces L(+) lactic acid(dextrorotatory) under homo-fermentation conditions. It has beenisolated from natural sources, such as heat-treated soil samplesinoculated into nutrient medium (see e.g., Bergey's Manual of SystemicBacteriology, Vol. 2, Sneath, P. H. A. et al., eds., Williams & Wilkins,Baltimore, Md., 1986). Purified Bacillus coagulans strains have servedas a source of enzymes including endonucleases (e.g., U.S. Pat. No.5,200,336); amylase (U.S. Pat. No. 4,980,180); lactase (U.S. Pat. No.4,323,651) and cyclo-malto-dextrin glucano-transferase (U.S. Pat. No.5,102,800). Bacillus coagulans has also been utilized to produce lacticacid (U.S. Pat. No. 5,079,164). A strain of Bacillus coagulans (alsoreferred to as Lactobacillus sporogenes; Sakaguti & Nakayama, ATCC No.31284) has been combined with other lactic acid producing bacteria andBacillus natto to produce a fermented food product from steamed soybeans(U.S. Pat. No. 4,110,477). Bacillus coagulans strains have also beenused as animal feeds additives for poultry and livestock to reducedisease and improve feed utilization and, therefore, to increase growthrate in the animals (International PCT Patent Applications No. WO9314187 and No. WO 9411492). In particular, Bacillus coagulans strainshave been used as general nutritional supplements and agents to controlconstipation and diarrhea in humans and animals.

Purified Bacillus coagulans bacteria utilized in the present inventionare available from the American Type Culture Collection (ATCC,Rockville, Md.) using the following accession numbers: Bacilluscoagulans Hammer NRS 727 (ATCC No. 11014); Bacillus coagulans Hammerstrain C (ATCC No. 11369); Bacillus coagulans Hammer (ATCC No. 31284);and Bacillus coagulans Hammer NCA 4259 (ATCC No. 15949). PurifiedBacillus coagulans bacteria are also available from the DeutscheSarumlung von Mikroorganismen und Zellkuturen GmbH (Braunschweig,Germany) using the following accession numbers: Bacillus coagulansHammer 1915 (DSM No. 2356); Bacillus coagulans Hammer 1915 (DSM No.2383, corresponds to ATCC No. 11014); Bacillus coagulans Hammer (DSM No.2384, corresponds to ATCC No. 11369); and Bacillus coagulans Hammer (DSMNo. 2385, corresponds to ATCC No. 15949). Bacillus coagulans bacteriacan also be obtained from commercial suppliers such as SabinsaCorporation (Piscataway, N.J.) or K.K. Fermentation (Kyoto, Japan).

Bacillus coagulans strains and their growth requirements have beendescribed previously (see e.g., Baker, D. et al, 1960. Can. J.Microbiol. 6: 557-563; Nakamura, H. et al, 1988. Int. J. Syst.Bacteriol. 38: 63-73. In addition, various strains of Bacillus coagulanscan also be isolated from natural sources (e.g., heat-treated soilsamples) using well-known procedures (see e.g., Bergey's Manual ofSystemic Bacteriology, Vol. 2, p. 1117, Sneath, P. H. A. et al., eds.,Williams & Wilkins, Baltimore, Md., 1986).

It should be noted that Bacillus coagulans had previously beenmis-characterized as a Lactobacillus in view of the fact that, asoriginally described, this bacterium was labeled as Lactobacillussporogenes (see Nakamura et al. 1988. Int. J. Syst. Bacteriol. 38:63-73). However, initial classification was incorrect due to the factthat Bacillus coagulans produces spores and through metabolism excretesL(+)-lactic acid, both aspects which provide key features to itsutility. Instead, these developmental and metabolic aspects requiredthat the bacterium be classified as a lactic acid bacillus, andtherefore it was re-designated. In addition, it is not generallyappreciated that classic Lactobacillus species are unsuitable forcolonization of the gut due to their instability in the harsh (i.e.,acidic) pH environment of the bile, particularly human bile. Incontrast, Bacillus coagulans is able to survive and colonize thegastrointestinal tract in the bile environment and even grown in thislow pH range. In particular, the human bile environment is differentfrom the bile environment of animal models, and heretofore there has notbeen any accurate descriptions of Bacillus coagulans growth in humangastrointestinal tract models.

2.2 Growth of Bacillus coagulans

Bacillus coagulans is aerobic and facultative, grown typically innutrient broth, pH 5.7 to 6.8, containing up to 2% (by wt) NaCl,although neither NaCl nor KCI are an absolute requirement for growth. ApH value ranging from approximately pH 4 to pH 6 is optimum forinitiation of sporulation. It is optimally grown at about 30° C. toabout 55° C., and the spores can withstand pasteurization. It exhibitsfacultative and heterotrophic growth by utilizing a nitrate or sulfatesource. Additional metabolic characteristics of Bacillus coagulans aresummarized in FIG. 1.

Bacillus coagulans can be grown in a variety of media, although it hasbeen found that certain growth conditions produce a culture which yieldsa high level of sporulation. For example, sporulation is enhanced if theculture medium includes 10 mg/liter of manganese sulfate, yielding aratio of spores to vegetative cells of about 80:20. In addition, certaingrowth conditions produce a bacterial spore which contains a spectrum ofmetabolic enzymes particularly suited for the present invention (i.e.,the control of microbial infections).

(A) Culture of Vegetative Bacillus coagulans

Bacillus coagulans is aerobic and facultative, and is typically culturedat pH 5.7 to 6.8, in a nutrient broth containing up to 2% (by wt) NaCl,although neither NaCl, nor KCl are required for growth. A pH of about4.0 to about 7.5 is optimum for initiation of sporulation (i.e., theformation of spores). The bacteria are optimally grown at 30° C. to 45°C., and the spores can withstand pasteurization. Additionally, thebacteria exhibit facultative and heterotrophic growth by utilizing anitrate or sulfate source.

Bacillus coagulans can be cultured in a variety of media, although ithas been demonstrated that certain growth conditions are moreefficacious at producing a culture which yields a high level ofsporulation. For example, sporulation is demonstrated to be enhanced ifthe culture medium includes 10 mg/l of MgSO₄ sulfate, yielding a ratioof spores to vegetative cells of approximately 80:20. In addition,certain culture conditions produce a bacterial spore which contains aspectrum of metabolic enzymes particularly suited for the presentinvention (i.e., production of lactic acid and enzymes for the enhancedprobiotic activity and biodegradation). Although the spores produced bythese aforementioned culture conditions are preferred, various othercompatible culture conditions which produce viable Bacillus coagulansspores may be utilized in the practice of the present invention.

Suitable media for the culture of Bacillus coagulans include: PDB(potato dextrose broth); TSB (tryptic soy broth); and NB (nutrientbroth), which are all well-known within the field and available from avariety of sources. In one embodiment of the present invention, mediasupplements which contain enzymatic digests of poultry and/or fishtissue, and containing food yeast are particularly preferred. Apreferred supplement produces a media containing at least 60% protein,and about 20% complex carbohydrates and 6% lipids. Media can be obtainedfrom a variety of commercial sources, notably DIFCO (Newark, N.J.); BBL(Cockeyesville, Md.); Advanced Microbial Systems (Shakopee, Minn.); andTroy Biologicals (Troy, Md.

In a preferred embodiment of the present invention, a culture ofBacillus coagulans Hammer bacteria (ATCC No. 31284) was inoculated andgrown to a cell density of approximately 1×10⁸ to 1×10⁹ cells/ml innutrient broth containing: 5.0 g Peptone; 3.0 g Meat Extract; 10-30 mgMnSO₄ and 1,000 ml distilled water, the broth was then adjusted to pH7.0. The bacteria were cultured by utilization of a standard airliftfermentation vessel at 30° C. The range of MnSO₄ acceptable forsporulation was found to be 1.0 mg/l to 1.0 g/l. The vegetativebacterial cells can actively reproduce up to 65° C., and the spores arestable up to 90° C.

2.3 Extracellular Products Possessing Anti-Microbial Activity

Bacillus coagulans cultures contain secreted products which possessanti-microbial activity. These secreted products are useful intherapeutic compositions according to the present invention. Cellcultures are harvested as described above, and the culture supernatantsare collected, by filtration or centrifugation, or both, and theresulting supernatant contains anti-microbial activity useful in thetherapeutic compositions of the present invention.

The preparation of a Bacillus coagulans extracellular products will bemore fully described in the Specific Examples section, infra.

(A) Preparation of B. coagulans and P. lindbergii Extracellular Products

One liter cultures of either Bacillus coagulans or Pseudomonaslindbergii were prepared as described supra, except that thefructo-oligosaccharide (FOS) was omitted. The culture was maintained for5 days as described, at which time FOS was added at a concentration of 5g/liter, and the culture was continued. Subsequently, 20 ml of carrotpulp was then added at day 7, and the culture was harvested when theculture became saturated (i.e., no substantial cell division).

The culture was first autoclaved for 30 minutes at 250° F., and thencentrifuged at 4000 r.p.m. for 15 mm. The resulting supernatant wascollected and filtered in a Buchner funnel through a 0.8 μm filter. Thefiltrate was collected and further filtered through a 0.2 μm Nalgevacuum filter. The resulting final filtrate was then collected (anapproximate volume of 900 ml) to form a liquid containing anextracellular product which was to be quantitatively analyzed andutilized in the subsequent inhibition studies.

The results of these aforementioned analytical methodologiesdemonstrated that the culture supernatants from both Bacillus coagulansand Pseudomonas lindbergii are very heterogeneous in nature, containinga plurality of proteinaceous and organic molecules. However, themolecules which predominate are the proteins, of which there are a totalof 20 distinct species in each of the samples. These protein species canbe further fractionated by use of ion exchange chromatography, thusallowing additional characterization. Furthermore, there are alsonumerous pigmented molecules (i.e., molecules which absorb visiblelight) that are both highly conjugated (based upon their absorbance athigh wavelengths) and hydrophobic (based upon their preference fornon-polar solvents and retention on the C-18 HPLC column).

Following the aforementioned analysis and characterization, 1 ml of theaforementioned extracellular product was added to the test plate inplace of the bacterium. After an identical culture time, a zone ofinhibition of approximately 10 to 25 mm in diameter was observed. Asdesignated herein, “excellent inhibition” means the zone was 10 mm orgreater in diameter; and “good inhibition” means the zone was greaterthan 2 mm in diameter but less than 10 mm in diameter. Thus, theseresults illustrate the potent anti-microbial activity of the Bacilluscoagulans extracellular product, which is of “excellent” quality usingthe terminology set forth above.

In an additional assay, a comparison of the anti-mycotic, Fluconazolewith Bacillus coagulans supernatant in the inhibition of variousbacterial, fungal, and yeast species, was performed. As illustrated inFIG. 2, these supernatants were effective in inhibiting a majority ofthe organisms against which they were tested. Serial dilutions of theBacillus coagulans supernatant were performed with RPMI medium andinhibition was determined at 80% in accordance with the NCCLS standardfor anti-fungal susceptibility.

Specifically, the results demonstrated that Trichophyton rubrum wastotally inhibited by undiluted supernatant, and 1:2, 1:4, 1:8, 1:16,1:32, 1:64, 1:128, and 1:256 serial dilutions, and the organism was 80%inhibited by the compound diluted 1:512 with RPMI medium. Trichophytonmentagrophytes was totally inhibited by the undiluted supernatant, and1:2, 1:4, 1:8, and 1:16 serial dilutions, and the organism was 80%inhibited by the supernatant diluted 1:32 with RPMI medium. Candidaparapsilosis was totally inhibited by the undiluted supernatant and 1:2,1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256 serial dilutions, and theorganism was 80% inhibited by the supernatant diluted 1:16 with RPMImedium. Candida albicans was totally inhibited by the undilutedsupernatant and a 1:2 dilution, and the organism was 80% inhibited bythe supernatant diluted 1:4 with RPMI medium. Acremonium sp. was totallyinhibited by the undiluted supernatant and was 80% inhibited by thesupernatant diluted 1:2 with RPMI medium. Scopulariopis sp. was 80%inhibited by the undiluted supernatant, but was uninhibited by any ofthe serial dilutions of the supernatant. The supernatant showed noinhibitory activity against Candida glabrata, Candida krusel, or the twoAspergillus species. Thus, the supernatant was demonstrated to possessmarked inhibitory activity, in a wide variety of dilutions, against amajority of the tested organisms. Moreover, the Bacillus coagulanssupernatant appeared to be extremely effective against dermatophytes(e.g. Trichophyton sp.), which are a causative organism in manymammalian dermal diseases.

In a preferred embodiment of the present invention, the liquidcontaining the extracellular product was formulated into a liquidointment composition for use in direct application onto a tissue using adropper, such as would be convenient to treat a fungal infection of thetoe nail. This liquid ointment was prepared by combining the liquidextracellular product produced above with Emu Oil in a ratio ofapproximately 8:2, and trace fragrances were added to produce anaesthetic component.

Alternatively, one may use any liposomal or oil based transdermaldelivery component in place of the Emu Oil. The typical ratio ofprobiotic extracellular product to carrier or delivery component is arange of from approximately 1% to 90% probiotic, and preferably isapproximately 10% to 75% probiotic.

2.4 Anti-Microbial Activity of Bacillus coagulans

It is well-documented clinically that many species of bacterial, mycoticand yeast pathogens possess the ability to cause a variety of disorders.Therefore, the utilization of the probiotic microorganism-containingcompositions of the present invention inhibits these pathogens areuseful in the prophylactic or therapeutic treatment of conditionsassociated with infection by these aforementioned pathogens.

Pathogenic bacteria inhibited by Bacillus coagulans activity include,for example, Staphylococcus aureus, Staphylococcus epidermidus,Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli (i.e.,entero-hemorragic species), numerous Clostridium species (e.g.,Clostridium perfingens, Clostridium botulinum, Clostridium tributrycum,Clostridium sporogenes, and the like); Gardnereia vaginails;Proponbacterium acnes; Aeromonas hydrophia; Aspergillus species; Proteusspecies; and Klebsiella species.

Pathogenic yeast and other fungi inhibited by Bacillus coagulansactivity include Candida albicans, Candida tropicalis and Trichophytonmentagrophytes, Trichophyton interdigitale, Trichophyton rubrum, andTrichophyton yaoundei.

Bacillus coagulans has also been demonstrated to inhibit Herpes simplexviruses I (HSV-I; oral “cold sores” and Herpetic Whitlow) and Herpessimplex II (HSV-II; genital herpes) and Herpes zoster (shingles)infections.

These aforementioned pathogens have been associated with a variety ofdisorders including, but not limited to: diaper rash; oral, genital,cervical and vaginal yeast infections; toxic shock syndrome; chronicmucocutaneous candidaiasis; dermatophytosis; bacterial vaginosis; tinealfungal infections (e.g., ringworm, athlete's foot, and jock itch); scalpand nail fungal infections; superficial skin disorders (e.g.,erysipelas, open-wound infections, acne, abscess, boil, eczema,dermatitis, contact dermatitis, hypersensitinitis, contact lesions, bedsores, and diabetic lesions); miscellaneous opportunistic infections;oral and genital viral lesions, and the like conditions as are wellknown in the art. Therefore, topical use of compositions containing theBacillus coagulans active agents that inhibit these pathogens are usefulin preventing or treating these conditions.

The various pathogens, which may be treated by use of the therapeuticcompositions of the present invention, and their associated disordersare illustrated in FIG. 3. It should be noted, however, that thepathogens listed in FIG. 3 are set forth as examples only, and are notmeant to be limiting to the types of organisms which can be treated byuse of the methodologies or compositions of the present invention.Accordingly, various other skin- and mucous membrane-infecting microbesand dermatophytes can also be treated by use of the present compositionsand methods disclosed herein.

The aforementioned anti-microbial activity of a therapeutic compositionof the present invention will be more fully-described in the SpecificExamples section, infra.

(A) Anti-Mycotic Activity of Bacillus coagulans

The ability of Bacillus coagulans to inhibit various fungal pathogenswas demonstrated using an in vitro assay. In the assay, potato-dextroseplates (DIFCO®, Detroit, Mich.) were prepared using standard proceduresand were inoculated individually with a confluent bed (about 1.7×10⁶) ofvarious species of the fungus Trichophyton. The tested fungal strains ofTrichophyton species (available from the American Type CultureCollection (ATCC; Rockville, Md.)) and their ATCC accession numbers, aswell as the results of in vitro inhibition by Bacillus coagulans areillustrated in FIG. 4.

Inhibition by Bacillus coagulans was ascertained by placing on the plateapproximately 1.5×10⁶ colony forming units (CFU) in 10 μl of broth orbuffer, plated directly in the center of the potato-dextrose plate, withone test locus per plate. The size of each test locus was approximately8 mm in diameter and a minimum of three tests were performed for eachinhibition assay. The negative control consisted of a 10 ml volume ofsterile saline solution, whereas the positive control consisted of a 10ml volume 2% Miconazole(1-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxylmethyl-1,11-imidazolewithin an inert cream.

The plates were then incubated for approximately 18 hr at 30° C., atwhich time the zones of inhibition were measured. As designated herein,“excellent inhibition” means the zone was 10 mm or greater in diameter;and “good inhibition” means the zone was greater than 2 mm in diameter,but less than 10 mm in diameter.

The results of the in vitro inhibition by Bacillus coagulans isillustrated in FIG. 4. For each of the Trichophyton species tested, thedisease condition associated with an infection is indicated in column 2of FIG. 4. For comparison, no zone of inhibition was seen with thenegative control, whereas good inhibition (approximately 8.5 mmdiameter, mean average of three tests) was seen with the positivecontrol.

In one embodiment of the present invention, the extracellular product ofthe selected Bacillus strain (preferably Bacillus coagulans) and/orPseudomonas lindbergii is combined with a therapeutically-effective doseof an anti-fungal agent and Emu Oil. In preferred embodiments of thepresent invention, the extracellular product of the aforementionedlactic acid-producing bacterial strains is combined with a therapeuticconcentration of one or more anti-fungal agents, including, but notlimited to: Dapsone, Fluconazole, Flucytosine, Griseofulvin,Itraconazole, Ketoconazole, Miconazole KI, Amphotericin B,Carbol-Fuchsin, Ciclopirox, Clotrimzole, Econazole, Haloprogin,Mafenide, Miconazole, Naftifine, Nystatin, Oxiconazole, SilverSulfadiazine, Sulconazole, Terbinafine, Tioconazole, Tolnafiate,Undecylenic acid, Butoconazle, Clotrimazole, Econazole, Gentian Violet,Miconazole, Nystatin, Terconazole, and Tioconazole.

(B) Inhibition of Yeast by Bacillus coagulans

Similarly, the ability of Bacillus coagulans to inhibit various yeastpathogens was demonstrated in vitro for four species of Candida, all ofwhich are available from the American Type Culture Collection (ATCC;Rockville, Md.). Each of the yeast pathogens and their ATCC accessionnumbers are illustrated in FIG. 5.

In the in vitro inhibition assay, potato-dextrose plates (DIFCO®,Detroit, Mich.) were prepared using standard procedures and wereinoculated individually with a confluent bed about 1.7×10⁶ of the fourspecies of Candida. Inhibition by Bacillus coagulans was tested byplacing on the plate about 1.5×10⁶ colony forming units (CFU) in 10 μlof broth or buffer, plated directly in the center of the potato-dextroseplate with one test locus of about 8 mm in diameter per plate. A minimumof three tests were performed for each inhibition assay. The negativecontrol consisted of a 10 μl volume of a sterile saline solution,whereas the positive control consisted of a 1 μl volume of Miconazolecream.

The plates were then incubated for approximately 18 hr at 30° C., atwhich time the zones of inhibition were measured. As designated herein,“excellent inhibition” means the zone was 10 mm or greater in diameter;and “good inhibition” means the zone was greater than 2 mm in diameter,but less than 10 mm in diameter.

The results of the in vitro tests are shown in FIG. 5 with thepathological conditions in humans associated with infection by theCandida species shown in column 2. As expected, no inhibition was seenwith the negative control and good inhibition (approximately 8.7 mmdiameter; average of three tests) was seen with the positive control.

(C) Anti-Microbial Activity of Bacillus coagulans

The ability of Bacillus coagulans to inhibit various opportunisticbacterial pathogens was quantitatively ascertained by use of an in vitroassay. This assay is part of a standardized bacterial pathogen screen(developed by the U.S. Food and Drug Administration(FDA)) and iscommercially available on solid support disks (DIFCO® BACTROL® DiskSet). To perform the assay, potato-dextrose plates (DIFCO®) wereinitially prepared using standard procedures. The plates were thenindividually inoculated with each of the bacteria (approximately 1.5×10⁶CFU) to be tested, so as to form a confluent bacterial bed.

Inhibition by Bacillus coagulans was subsequently ascertained by placingapproximately 1.5×10⁶ CFU of Bacillus coagulans in 10 μl of broth orbuffer, directly in the center of the potato-dextrose plate, with onetest locus being approximately 8 mm in diameter per plate. A minimum ofthree test loci were used for each assay. The negative control consistedof a 10 μl volume of a sterile saline solution, whereas the positivecontrol consisted of a 10 μl volume of glutaraldehyde. The plates werethen incubated for approximately about 18 hr at 30° C., at which timethe zones of inhibition were measured. As designated herein, “excellentinhibition” means the zone was 10 mm or greater in diameter; and “goodinhibition” means the zone was greater than 2 mm in diameter but lessthan 10 mm in diameter.

As expected, no “inhibition” was seen with the negative, saline control,and excellent “inhibition” (approximately 16.2 mm diameter; average ofthree tests) was seen with the positive, glutaraldehyde control. For theenteric microorganisms tested, the following inhibition by Bacilluscoagulans was found: (i) Clostridium species—excellent inhibition; (ii)Escherichia coli—excellent inhibition; (iii) Clostridiumspecies—excellent inhibition, where the zone of inhibition wasconsistently greater than 15 mm in diameter. Similarly, excellentinhibition was also seen for the opportunistic pathogens Pseudomonasaeruginosa, and Staphylococcus aereus.

In summation, pathogenic enteric bacteria which were shown to beinhibited by Bacillus coagulans activity include, but are not limitedto: Staphylococcus aureus; Staphylococcus epidermidus; Streptococcuspyogenes; Pseudomonas aeruginosa; Escherichia coli (enterohemorragicspecies); numerous Clostridium species (e.g., Clostridium perfingens,Clostridium botulinum, Clostridium tributrycum, Clostridium sporogenes,and the like); Gardnereia vaginails; Proponbacterium acnes; Aeromonashydrophia; Aspergillus species; Proteus species; and Klebsiella species.

In one embodiment of the present invention, the extracellular product ofthe selected Bacillus strain (preferably Bacillus coagulans) and/orPseudomonas lindbergii is combined with a therapeutically-effective doseof an antibiotic and Emu Oil. In preferred embodiments of the presentinvention, the extracellular product of the aforementioned lacticacid-producing bacterial strains is combined with a therapeuticconcentration of one or more antibiotics, including, but not limited to:Gentamicin; Vancomycin; Oxacillin; Tetracyclines; Nitroflurantoin;Chloramphenicol; Clindamycin; Trimethoprim-sulfamethoxasole; a member ofthe Cephlosporin antibiotic family (e.g., Cefaclor, Cefadroxil,Cefixime, Cefprozil, Ceftriaxone, Cefuroxime, Cephalexin, Loracarbef,and the like); a member of the Penicillin family of antibiotics (e.g.,Ampicillin, Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin,Penicillin VK, and the like); with a member of the Fluoroquinolonefamily of antibiotics (e.g., Ciprofloxacin, Grepafloxacin, Levofloxacin,Lomefloxacin, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, andthe like); or a member of the Macrolide antibiotic family (e.g.,Azithromycin, Erythromycin, and the like).

3. Therapeutic Compositions

Compositions of the present invention which are suitable for use inpreventing, treating, and/or controlling microbial infections comprisean active ingredient, specifically a Bacillus coagulans supernatant orfiltrate of a Bacillus coagulans or Pseudomonas lindbergii culture grownto saturation and Emu Oil.

The therapeutic compositions may also include, but are not limited tothe inclusion of: known antioxidants (e.g., vitamin E); bufferingagents; lubricants (e.g., synthetic or natural beeswax); sunscreens(e.g., para-aminobenzoic acid); and other cosmetic agents (e.g.,coloring agents, fragrances, oils, essential oils, moisturizers ordrying agents). Thickening agents (e.g., polyvinylpyrrolidone,polyethylene glycol or carboxymethyicellulose) may also be added to thecompositions.

In the therapeutic compositions of the present invention, the activeagents are combined with a “carrier” which is physiologically compatiblewith the skin, membrane, or mucosal tissue of a human or animal to whichit is topically administered. Specifically, in the preferred embodiment,the carrier is substantially inactive, with the exception of itsintrinsic surfactant properties which are used in the production of asuspension of the active ingredients. The compositions may include otherphysiologically active constituents that do not interfere with theefficacy of the active agents in the composition. The carriers utilizedin the therapeutic compositions of the present invention are preferablyliquid or gel-based materials for use in liquid or gel formulations. Thespecific formulations depend, in part, upon the routes or modes ofadministration. Suitable liquid or gel-based carriers are well-known inthe art (e.g., water, physiological salt solutions, urea, methanol,ethanol, propanol, butanol, ethylene glycol and propylene glycol, andthe like). Preferably, water-based carriers are approximately neutralpH.

Suitable carriers include aqueous and oleaginous carries such as, forexample, white petrolatum, isopropyl myristate, lanolin or lanolinalcohols, mineral oil, fragrant or essential oil, nasturtium extractoil, sorbitan mono-oleate, propylene glycol, cetylstearyl alcohol(together or in various combinations), hydroxypropyl cellulose(MW=100,000 to 1,000,000), detergents (e.g., polyoxyl stearate or sodiumlauryl sulfate) and mixed with water to form a lotion, gel, cream orsemi-solid composition. Other suitable carriers comprise water-in-oil oroil-in-water emulsions and mixtures of emulsifiers and emollients withsolvents such as sucrose stearate, sucrose cocoate, sucrose distearate,mineral oil, propylene glycol, 2-ethyl-1,3-hexanediol,polyoxypropylene-15-stearyl ether and water. For example, emulsionscontaining water, glycerol stearate, glycerin, mineral oil, syntheticspermaceti, cetyl alcohol, butylparaben, propylparaben and methylparabenare commercially available. Preservatives may also be included in thecarrier including methylparaben, propylparaben, benzyl alcohol andethylene diamine tetraacetate salts. The composition may also include aplasticizer such as glycerol or polyethylene glycol (MW 400 to 20,000).The composition of the carrier can be varied so long as it does notinterfere significantly with the pharmacological activity of the activeingredients of the therapeutic composition.

A therapeutic composition of the present invention may be formulated tobe suitable for application in a variety of manners, for example, in acream for topical application to the skin (e.g., for ringworm orathlete's foot), in a liquid for finger or toe nails (e.g., for tineapedis), and the like. Other formulations will be readily apparent to oneskilled in the art and will be discussed more fully in the SpecificExamples section, infra.

3.1 The Utilization of Emu Oil as a Carrier in Therapeutic Compositions

As previously discussed supra, numerous animal-derived lipids have beenexamined for utilization as “carrying agents”, which are used todisperse and facilitate penetration of these therapeutic compositionsthrough the various dermal and cuticular membranes and tissues. However,prior to the disclosure contained herein, there has been little successin finding an agent that is able to penetrate dense cuticular materialsuch as finger/toenails and animal hooves.

Disclosed herein is the use of an animal-derived lipid, Emu Oil, as a“carrying agent” to facilitate the dispersion and penetration of thetherapeutic compositions of the present invention through the variousdermal and cuticular membranes and tissues, and has been demonstrated tomarkedly increase the efficacy of anti-microbial and anti-fungaltherapies. This lipid material is extracted from the Emu (DromaisNovae-Hollandiae), an indigenous bird of Australia and New Zealand.Although Emu Oil has been previously described, the uses which aredetailed in these documents elaborate only its benefits as ananti-inflammatory agent for arthritis and its uses for cardiovascularhealth when ingested, which is similar to the use of Omega-3 fish oilsto improve high-density lipoprotein (HDL) cholesterol.

Accordingly, both human and animal dermal diseases, caused by bacterialand/or mycotic dermatophytes, can be mitigated or prevented, whileconcomitantly maintaining dermal and cuticular health, by use of acombination of active agents in a therapeutic composition which includesanti-fungal/anti-bacterial agents (e.g., organic molecules, proteins andcarbohydrates and/or bacterial fermentation products) in combinationwith Emu Oil. In a preferred embodiment of the present invention, atherapeutically-effective concentration of Emu Oil is combined with thefermentation products of bacteria that have been shown to produceinhibitory metabolites (e.g., Bacillus coagulans) and, optionally, withan anti-microbial agent (e.g., an anti-fungal or antibiotic), in apharmaceutically-acceptable carrier suitable for administration to thedermal and/or cuticular membranes of an animal.

In one embodiment of the bacterial supernatant composition, thebacterial strain is a member of the Lactobacillus genus including, butnot limited to: Lactobacillus acidophilus, Lactobacillus plantarum,Lactobacillus salivarius, Lactobacillus delbrukil, Lactobacillusrhamnosus, Lactobacillus bulgaricus, Lactobacillus gaserli,Lactobacillus jensenii and Lactobacillus sporogenes. In anotherembodiment, the bacterial strain is a member of the genus Enterococccus,which include, but are not limited to: Bacillus facium and Enterococccusthermophilus. In another embodiment, the bacterial strain is a member ofthe Bifidiobacterium genus, which include, but are not limited to:Bacillus longum, Bacillus infantis, Bacillus bifidus, and Bacillusbifidum. In another embodiment, the bacterial strain is a member of thegenus Bacillus, which include, but are not limited to: Bacilluscoagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillussubtilis, Bacillus megaterium, Bacillus licheniformis, Bacillusmycoides, Bacillus pumilus, Bacillus lentus, Bacillus uniflagellatus,Bacillus cereus and Bacillus circulans. In another embodiment thebacterial strain is a member of the genus Pseudomonas, which include,but are not limited to: Pseudomonas aeruginosa, Pseudomonas putida,Pseudomonas lindbergii, Pseudomonas cepacia, Pseudomonas florescenes,and Pseudomonas 679-2. In another embodiment of the present, the strainis a member of the genus Sporolactobacillus. In various otherembodiments of the present invention, the bacterial strains which areutilized are members of the genus Micromonospora, Sporolactobacillus,Micrococcus, Berkholderia, Rhodococcus and any of the other bacteriawhich possess the ability to produce a metabolite that hasanti-bacterial, anti-mycotic, or anti-viral activity.

In other embodiments of the present invention, the aforementionedbacterial supernatant compositions may be combined with an activeanti-microbial agent which is a non-microbially-derived compound. Thesenon-microbially-derived, anti-microbial compound may include, but arenot limited to: a quartenary ammonium chloride, an iodine or iodifercompound (e.g., Betadine™), a phenolic compound, an alcohol compound ortincture (e.g., ethanol, isopropyl, and the like). In other embodiments,the non-microbially-derived, anti-microbial compound is a systemicanti-fungal compound, including, but not limited to: Amphotericin B,Dapsone, Fluconazole, Flucytosine, Griseofulvin, Itraconazole,Kietoconazole, or Miconazole KI. In other embodiments, thenon-microbially-derived, anti-microbial compound is a topicalanti-fungal compound, including, but not limited to: Amphotericin B,Carbol-Fuchsin, Ciclopirox, Clotrimzole, Econazole, Haloprogin,Ketoconazole, Mafenide, Miconazole, Naftifine, Nystatin, Oxiconazole,Silver Sulfadiazine, Sulconazole, Terbinafine, Tioconazole, Tolnafiate,or Undecylenic acid. In other embodiments, the non-microbially-derived,anti-microbial compound is an anti-fungal vaginal compound, including,but not limited to: Butoconazle, Clotrimazole, Econazole, GentianViolet, Miconazole, Nystatin, Terconazole, or Tioconazole.

Specific methods for the utilization of Emu Oil-containing therapeuticcompositions will be more fully described in the Specific Examplessection, infra.

3.2 Therapeutic Methods for Treatment of Microbial Infections

The present invention discloses methodologies for treating, reducing,and/or controlling microbial infections in a variety of skin and mucosalmembrane tissues using a therapeutic composition or therapeutic articleof manufacture of this invention. Optimally the compositions effectivelyreduce the bacterial, yeast, fungal and/or viral titer in the treatedindividual, particularly at the site of application of the topicalcomposition. For example, the pathogenic microbial titer in lesions hasbeen demonstrated to be significantly reduced following the topicaladministration of the therapeutic composition of the present inventionto the affected area(s) of the skin or mucous membrane. The disclosedmethods of treatment also reduce symptoms of pathogenic microbialinfection (e.g., pain associated with infected or microbial-causedlesions) and promote more rapid healing than would be found without saidtreatment.

The method of the present invention includes administration of acomposition containing the active Bacillus ingredient to a human oranimal to treat or prevent microbial (i.e., bacterial, yeast, fungal orviral) infection. Administration is preferably to the skin or a mucousmembrane using a cream, lotion, gel, oil, ointment, suspension, aerosolspray, powder, semi-solid formulation (e.g., a suppository), or articleof manufacture, all formulated so as to contain a therapeuticcomposition of the present invention using methods well-known in theart.

Application of the therapeutic composition of the present invention,containing the active agent effective in preventing or treating amicrobial infection, generally consists of one to ten applications of acomposition for a time period of one day up to one month. Applicationsare generally once every twelve hours and up to once every four hours.Preferably, two to four applications of the therapeutic composition perday, for one to seven days are sufficient to prevent or treat amicrobial infection. For topical applications, the therapeuticcompositions are preferably applied to lesions daily as soon assymptomology (e.g., pain, swelling or inflammation) is detected. Thespecific route, dosage, and timing of the administration will depend, inpart, on the particular pathogen and/or condition being treated, as wellas the extent of the condition.

Specific methods for the treatment of microbial infections will be morefully described in the Specific Examples section, infra, and include,but are not limited to, the treatment of diaper rash, vaginal yeastinfection, opportunistic skin infection, meal fungal infection,superficial skin infection, acne, cold sores, genital Herpes lesions,Herpetic Whitlow, shingles, athlete's foot, and the like.

3.3 Therapeutic Systems for Treatment of Microbial Infections

The present invention further discloses a therapeutic system fortreating, reducing, and/or controlling microbial infections comprising acontainer containing a label and a therapeutic composition of thepresent invention, wherein said label comprises instructions for the useof the therapeutic composition for the treatment of the infection.

For example, the therapeutic system can comprise one or more unitdosages of a therapeutic composition of the present invention.Alternatively, the system can contain bulk quantities of the therapeuticcomposition. The label contains instructions for using the therapeuticcomposition in either unit dose or in bulk forms as appropriate, and mayinclude information regarding storage of the composition, diseaseindications, dosages, routes and modes of administration and the likeinformation.

4. Articles of Manufacture

The present invention also discloses various articles of manufacturewhich utilize the beneficial aspects of the present invention bycombination of the therapeutic composition with various medical orpersonal hygiene devices so as to reduce or prevent microbial infectionsassociated with the use of these devices. The articles comprisecompositions of an extracellular product of a lactic acid-producingbacterial species, Emu Oil, and, optionally, an anti-microbial agentapplied to a solid surface or impregnated into a solid matrix of anydevice or article of manufacture that is intended to be in contact withskin or a mucous membrane. Preferably the solid surface is a flexiblearticle than can be worn on or wiped on the skin or mucous membrane.More preferably, when the flexible item carrying the active agent is tobe worn on the skin it includes a means for attaching the article to theskin such as, for example, an adhesive layer, inter-engaging hook andpile (i.e., Velcro®) connectors, or other well-known means of attachmentsuch as ties, snap closures, elastic, buttons and the like.

Specific embodiments which include a Bacillus and/or isolated Bacilluscoagulans active agent are diapers, towelettes (e.g., baby wipes orfeminine hygiene towelettes), tampons, dermal patches, adhesive tape,absorbent pads, articles of clothing (e.g., underclothes, sleepingapparel), bath towels, wash cloths, and the like. The article may bemade of fibrous woven, knitted or non-woven materials, occlusive ornon-exclusive films or membranes, synthetic polymer fibers, films,membranes and foams (e.g., nylon, polytetrafluoroethylene (PTFE, such asTeflon® or Gore-Tex®), polystyrene, polycarbonate, polyvinylchloride andpolysulphone). All of these forms are well-known within the art andinclude, for example, knitted or woven fabrics, non-woven fabrics suchas felt and batting, fiber balls of cotton, rayon, cellulose orsynthetic fibers and the like materials.

The Bacillus and/or Bacillus coagulans isolated active agent can beapplied to the solid surface using any of a variety of known methodsincluding, for example, applying a powder, spray drying the probioticonto the material or soaking the material in a solution containing theprobiotic and then using the wetted material or drying the materialprior to use. Porous material may contain the Bacillus and/or theisolated active agent in the pores or interstices of the solid material.The Bacillus and/or the isolated active agent can be attached byadhesion, such as by attachment to an adhesive layer that is thenapplied to the skin (e.g., in a bandage or dermal patch). The Bacillusand/or the isolated active agent can be impregnated into the solidmaterial during the manufacturing process of the flexible article (e.g.,added to a synthetic composition before or during the polymerizationprocess). The pressure and heat resistance of Bacillus spores makes themparticularly suitable for incorporation into the material duringmanufacturing. Any of the solid materials carrying Bacillus and/or theisolated active agent can also be packaged individually or in groups,suitable for holding the treated material using standard packagingmaterials (e.g., in a shrink wrapper, sealed packet, protective wrapperor dispensing container suitable for holding dry or wet materials). Thearticle of manufacture can have applied thereon any of theadditional/optional components of a therapeutic composition of thisinvention, including carriers, salts, FOS, fragrances, and the like.

Any of a variety of methods for placing the therapeutic composition ontoa subject article can be used, and therefore the invention need not beso limited. However, preferred methods include a “spray-dry” method inwhich the material is exposed in a low humidity chamber to an atomizedmix containing a liquid composition, where the chamber is subsequentlyexposed to approximately 80-110° F. to dry the liquid, therebyimpregnating the material of the article with the components of thecomposition.

A typical concentration is from approximately 1×10⁵ to 1×10⁹ CFU ofviable bacterium or spores/in² of external surface of fibrouscarrier/article material. Following drying, the article is ready forstorage in a sterile package, or for direct use.

5. Specific Examples

The following examples relating to the present invention areillustrative and should not be construed as specifically limiting theinvention. Moreover, such variations of the invention, now known orlater developed, which would be within the purview of one skilled in theart are to be considered to fall within the scope of the presentinvention hereinafter claimed.

5.1 Treatment of Bacterial and Fungal Infections of the Dermis andCuticle

As previously discussed, various lactic acid-producing bacteria (e.g.,Bacillus coagulans and Pseudomonas lindbergii) have been shown toproduce extracellular products that are anti-fungal in nature althoughall of the products that have come from these bacteria are a result ofthe purification of a specific active analog such as a protein,carbohydrate or organic molecule to form a new anti-fungal compound. Ithas been suggested that the use of a single active agent contributes toresistant species of pathogenic fungi and as a result new generations ofanti-fungal compounds must be discovered in order to control these newdeveloping species. However, the use of a bacterial supernatant in itscrude or in a semi-refined state my be more effective in topicalapplications and may, in fact, decrease the rate of anti-fungalresistance by providing a more complex killing mechanism that is moredifficult to overcome than a single chemical agent or analog.

The use of Emu Oil as a “carrier” in the therapeutic compositions of thepresent invention markedly enhances efficacy in the prevention and/ortherapeutic treatment of fungal or bacterial infections of the dermisand cuticle in both humans and animals. These therapeutic compositionsare comprised of the fermentation products of specific bacterial strainsand, optionally, a commercially available antibiotic or anti-fungalagent in combination with an effective amount of Emu Oil in apharmaceutically acceptable cater suitable for administration to thedermal and/or cuticular membranes of a human or animal.

In various embodiments of the present invention, the final form of thetherapeutic composition may include, but is not limited to: a stabilizedgel, a lotion, a cream, a semi-solid roll-on stick, a fluid, an aerosol,a spray powder, or an emulsion.

The overall efficacy of the therapeutic compositions of the presentinvention is relative to the concentration of Emu Oil which is utilizedin the formulation. Specifically, it has been observed that higherpercentages of Emu Oil is more effective than lower percentages. Not tobe bound by any efficacious percentage, the range of Emu Oil used in atopical therapeutic composition of the present invention ranges fromapproximately 0.5% to 99.9%, with a more preferable range being betweenapproximately 10% to 75%, and the most preferable range being betweenapproximately 25% to 60%. The 0.5% to 99.9% ultimate effective range forEmu Oil concentration is due to the very small concentrations ofanti-microbial compounds which are typically used in the therapeuticcompositions of the present invention. For example, in a dermalapplication, the anti-fungal agent, Miconazole Nitrate, generallycomprises only 2% of the total formulation.

In another embodiment of the present invention, the extracellularproduct of the selected Bacillus strain (preferably Bacillus coagulans)and/or Pseudomonas lindbergii is combined with atherapeutically-effective dose of an anti-fungal agent and Emu Oil. Inpreferred embodiments of the present invention, the extracellularproduct of the aforementioned lactic acid-producing bacterial strains iscombined with a therapeutic concentration of one or more anti-fungalagents, including, but not limited to: Dapsone, Fluconazole,Flucytosine, Griseofulvin, Itraconazole, Ketoconazole, Miconazole KI,Amphotericin B, Carbol-Fuchsin, Ciclopirox, Clotrimzole, Econazole,Haloprogin, Mafenide, Miconazole, Naftifine, Nystatin, Oxiconazole,Silver Sulfadiazine, Sulconazole, Terbinafine, Tioconazole, Tolnafiate,Undecylenic acid, Butoconazle, Clotrimazole, Econazole, Gentian Violet,Miconazole, Nystatin, Terconazole, and Tioconazole.

In another embodiment of the present invention, the extracellularproduct of the selected Bacillus strain (preferably Bacillus coagulans)and/or Pseudomonas lindbergii is combined with atherapeutically-effective dose of an antibiotic and Emu Oil. Inpreferred embodiments of the present invention, the extracellularproduct of the aforementioned lactic acid-producing bacterial strains iscombined with a therapeutic concentration of one or more antibiotics,including, but not limited to: Gentamicin; Vancomycin; Oxacillin;Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-sulfamethoxasole; a member of the Cephlosporin antibioticfamily (e.g., Cefaclor, Cefadroxil, Cefixime, Cefprozil, Ceftriaxone,Cefuroxime, Cephalexin, Loracarbef, and the like); a member of thePenicillin family of antibiotics (e.g., Ampicillin,Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin, Penicillin VK, andthe like); with a member of the Fluoroquinolone family of antibiotics(e.g., Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); or amember of the Macrolide antibiotic family (e.g., Azithromycin,Erythromycin, and the like).

It should also be noted that the extracellular product of the selectedBacillus strain and/or Pseudomonas lindbergii and Emu Oil may becombined with antibiotic and anti-mycotic compounds within the sametherapeutic composition.

The following are examples of therapeutic compositions which have beendemonstrated to be effective in the mitigation of bacterial and mycoticdiseases of the dermis and cuticle.

Therapeutic Composition No. 1 Miconazole Nitrate, Fluconazole,Tolnaftate, 2% Ketoconazole or Intraconazole Emu Oil or Fraction Thereof90%  Emulsifier 5% Fragrance 3%

Therapeutic Composition No. 2 Quaternary Ammonium Chloride, Iodine, 10%Alcohol or Phenolic Compounds Emu Oil or Fraction Thereof 80% Emulsifier 7% Fragrance  3%

Therapeutic Composition No. 3 Bacterial Supernatant Composition 50%Fermentation Products Emu Oil or Fraction Thereof 40% Emulsifier  7%Fragrance  3%

Therapeutic Composition No. 4 Bacterial Supernatant Composition 50%Fermentation Products Emu Oil or Fraction Thereof 25% Lavender Oil  2%Hydrosperse Oil 20% Emulsifying Agents  3%

Therapeutic Composition No. 5 Antibiotic 2% Emu Oil or Fraction Thereof90%  Emulsifier 5% Fragrance 3%As previously discussed, these aforementioned therapeutic compositionsof the present invention may also be utilized in combination with otheranti-fungal and/or anti-microbial agents, as set forth, supra. Inaddition, various other materials (e.g., Titanium oxide) to enhance thewhitening of the toe or finger nail may also be used.

In a specific example, a therapeutic composition of the presentinvention, containing bacterial supernatant derived from Bacilluscoagulans, was used to mitigate the human fungal infection,Onychomycosis. One ml of the aforementioned therapeutic composition wasapplied after bathing to each infected nail. Treatment resulted in achange in the green-to-yellow color of the nail within 10 days, in allindividuals studied. In addition, within the first 7 days, the detritusunder the nail sloughed-off and the thickness of the nail (one of theclinical manifestations of the disease) began to subside. Although thetotal amount of time which was required to ameliorate this diseasevaried between each subject, the average time required ranged from onemonth for superficial infections to six months for more pronouncedOnychomycosis. Also, it must be taken into consideration that cosmeticappearance is an aspect of this disease that is independent of thepathology of the nail bed.

In has been demonstrated that the simultaneous anti-fungal action of thebacterial culture supernatant combined with the dermal-penetrating andhealing aspects of the Emu Oil work in a synergistic manner toameliorate the fungal infection. It is generally known that Emu Oilpossess the ability to rehydrate skin cells in a way that promotes thegrowth of new cells. Similarly, it is quite possible that Emu Oil actsin a similar manner in human nail and cuticular tissues.

In other specific examples, a therapeutic composition of the presentinvention, containing bacterial supernatant derived from Bacilluscoagulans, was also utilized to treat cases of diaper rash which werecomplicated with bacterial or fungal infections. Immediate (i.e.,approximately 18 hours) relief of the dermal inflammation and rednesswas achieved, and all of the infections were completely amelioratedwithin 48 hours. Similar results have been observed in the use of thesetherapeutic compositions in the treatment of Jock itch (Tinea cruris),Ringworm, Athlete's Foot (Tinea pedis), Scalp infections (Tineacapitis), Beard infections (Tinea barbae), Candidaiasis of the dermis,toe, fingernail and vulva, and other dermal and cuticular diseases.

Various equine hoof diseases (e.g., White Line disease, Hoof Thrush,Drop Sole, and even Clubbed Foot) have also responded to the use oftherapeutic compositions of the present invention, containing bacterialsupernatant derived from Bacillus coagulans, in the same manner asOnychomycosis in humans. In addition, similar to its physiologicalactivity in humans, Emu Oil may also function to rehydrate and stimulatenew cell growth within animal hooves and other cuticular materials.

5.2 Prophylactic or Therapeutic Treatment of Athlete's Foot

For the prevention or therapeutic treatment of athlete's foot (i.e.,tineal fungal infection), the -feet are washed with soap and water,dried thoroughly and a powder, cream, lotion, ointment or gel, such asthose described in the above examples is applied to the entire footarea. Preferably, the formulation includes approximately 0.5% to 20%Bacillus coagulans supernatant or filtrate of a Bacillus coagulans orPseudomonas lindbergii culture grown to saturation and 25% to 40% EMUOil (vol./vol.). Daily treatments are continued as needed.

Additionally, athlete's foot may be prevented or treated by using astandard insole insert (e.g., a fabric, fiber or synthetic foam) havingsprayed on the surface or impregnated therein with the Bacilluscoagulans or Pseudomonas lindbergii extracellular anti-fungal product.Such treated insoles may be worn daily for up to two to three months,after which they are discarded and replaced with fresh treated insoles.

5.3 Treatment of Tineal Fungal Infections

Ringworm (tinea versicolor) is caused by localized infections of theskin of the trunk and neck by dermatophyte fungus which colonizes theouter layer of the skin resulting in generally circular patches ofwhite, brown or pink flaking skin that are often itchy. Once ringworm isdetected, the affected area and a surrounding approximately 1 to 10 cm²area is treated twice daily with a cream or lotion containingapproximately 0.5% to 20% Bacillus coagulans supernatant or filtrate ofa Bacillus coagulans or Pseudomonas lindbergii culture grown tosaturation and 25% to 40% EMU Oil (vol./vol.).

For treatment of the related disorder, tinea cruris (i.e., “jock itch”),a cream, lotion, or aerosol spray containing approximately 0.5% to 20%Bacillus coagulans supernatant or filtrate of a Bacillus coagulans orPseudomonas lindbergii culture grown to saturation and 25% to 40% EMUOil (vol./vol.) is applied to the groin area to provide relief ofitching, chafing, burning rash and irritation. Treatment is twice daily,generally after bathing and at bedtime, until symptoms are no longerdetected.

Clothing, particularly underclothes and nightclothes that come incontact with the trunk and neck are sprayed with an aerosol containingapproximately 0.5% to 20% Bacillus coagulans supernatant or filtrate ofa Bacillus coagulans or Pseudomonas lindbergii culture grown tosaturation and 25% to 40% EMU Oil (vol./vol.), so as to ameliorate thespread of the infection to additional areas of the body.

5.4 Topical Application to Prevent Diaper Rash

An aerosol spray liquid containing the Bacillus coagulans or Pseudomonaslindbergii active, extracellular agent is applied to diapers by theconsumer before use. Alternatively, disposable diapers supplied from themanufacture may contain the Bacillus coagulans or Pseudomonas lindbergiiactive, extracellular agent impregnated into the diaper material whereit would be adjacent to the child's skin when in use. In both of theaforementioned embodiments, the composition utilized containsapproximately 0.5% to 20% Bacillus coagulans supernatant or filtrate ofa Bacillus coagulans or Pseudomonas lindbergii culture grown tosaturation and 25% to 40% EMU Oil (vol./vol.).

Alternatively or in addition to treating diapers with the Bacilluscoagulans or Pseudomonas lindbergii active, extracellular agent, thechild's skin in the diaper area can be treated with a saturated softcloth wipe, powder, aerosol spray liquid, aerosol spray powder, lotion,cream or ointment containing approximately 0.5% to 20% Bacilluscoagulans supernatant or filtrate of a Bacillus coagulans or Pseudomonaslindbergii culture grown to saturation and 25% to 40% EMU Oil(vol./vol.). Preferably, the aforementioned formulation is applied tothe child's skin after bathing and/or when the diapers are changed.

5.5 Topical Treatment of Vaginal Yeast Infection

(A) Vaginal Microecology

It is commonly known to those individuals skilled within the relevantarts that lactic acid-producing microorganisms (e.g., Lactobacillus)play an important role in the maintenance of a healthy vaginal ecology.However, the traditional methodologies utilized for the administrationof these biorational materials do not address the numerous modes ofinfection of Candida and Gardnerella species, which can cause seriousdisease.

The vast majority of gynecologists are adamant regarding the risks ofvaginal infections as a result of frequent bathing. Accordingly,gynecologists recommend the use of showers, rather than immersionbathing, to mitigate the probability of developing subsequent vaginalinfections due to the associated disturbances of the “normal,” lacticacid-producing vaginal flora.

(B) Yeast-Mediated Vaginal Infections

Yeast infections or vuvo-vaginal candidaiasis (VVC) is caused by variousspecies of Candida (e.g., primarily Candida albicans). Over 85% of allwomen, at one time or another, suffer from vuvo-vaginal candidaiasis.For example, the market within the United States market for anti-fungalcompounds which may be administered to ameliorate this disease is over$700 million dollars per year, with an associated 9-11% growth rate perannum. Moreover, each year, additional strains of these aforementionedmycotic pathogens are becoming resistant to the commonly utilizedanti-fungal compounds (e.g., Ketoconazole, Miconazole, Fluconazole, andthe like).

Healthy vaginal ecology is primarily dependant upon specific, indigenouslactic acid-producing microorganisms (e.g., Lactobacilli). Hence, therehave been numerous attempts within the prior art to develop productsand/or methodologies which will augment or re-establish these lacticacid-producing bacteria. For example, one product attempted to utilizehydrogen peroxide—(H₂O₂) producing Lactobacilli as a vaginal suppositorytherapy for the amelioration of vaginal yeast infections.

Viability of the microorganisms continues to be the main difficulty inthe use of Lactobacilli for vaginal supplementation, although it hasbeen suggested by many companies that market Lactobacilli vaginalsuppositories that any hardy bacterial strain is sufficient toaccomplish mycotic mitigation within the vagina. However, theseaforementioned companies primarily base their logic and subsequentassertions upon the fact that there are strains of Lactobacillus whichare able to colonize the vagina, and since their strain is a member ofthe genus Lactobacillus then it should be efficacious. Unfortunately,this supposition or deduction could not be more in error. In a recentstudy, which examined the various indigenous species and strains ofLactobacilli which colonized the vaginas of 100 healthy women. Theresults demonstrated that Lactobacillus acidophilus was not the mostcommon species of Lactobacillus isolated from the vaginas of thesewomen, but rather the most common strains were found to be:Lactobacillus jensenii; Lactobacillus gasserii; Lactobacillussalivarius; and Lactobacillus casel.

This aforementioned information, in combination with recent evidencewhich established that hydrogen peroxide (H₂O₂) is a mandatory metabolicby-product for effective bio-augmentation, disproves the previous beliefthat any strain of Lactobacillus is equally efficacious for use in asuppository-based administration format. Thus, these facts demonstratethe continued need for the development of a product for vaginalsupplementation, in combination with an efficacious method ofadministration, which ameliorates the potential physiological problemsassociated with the use of both bath products and bathing, in general.

(C) Bacterial-Mediated Vaginal Infections

Despite convincing evidence that lower reproductive tract infectionspossess the ability to migrate to the upper reproductive tract andproduce inflammation, stimulate premature labor, and the like, someclinicians still hold to the tenant that lower reproductive tractinfections and bacterial vaginosis are merely “markers” of upperreproductive tract infections.

It should be noted that bacterial vaginosis is not truly anmicroorganism-mediated infection, but instead a microecologic conditionin which there are dramatic alterations in the endogenous vaginalmicroflora. Specifically, bacterial vaginosis involves a reduction inthe overall number of lactic acid-producing bacterial strains, with aconcomitant multi-log population increase in a characteristic set ofmicroflora including, but not limited to: Gardnerella vaginalis, genitalanaerobes, and mycoplasmas. Interestingly, these latter microorganisms,along with Streptococci and Coliforms, are the same species as thosefound in chorioamnionitis.

Additionally, bacterial vaginosis is also associated with increasedconcentrations of bacterial endotoxin, proteases, mucinases, sialidases,IgA proteases, and phospholipases A2 and C in the lower reproductivetract. Both observational and interventional studies have shown that thepresence of bacterial vaginosis in the early stages of pregnancy isassociated with pre-term delivery and in later stages of gestation, withmiscarriage. These studies suggest that bacterial vaginosis is a directcause of adverse outcomes in pregnancy, rather than simply being asurrogate marker. Studies suggest that ascending infection or abnormallower reproductive tract microflora mediate adverse pregnancy outcomes.Similar microbe-host interactions occur in periodontal disease.

Bacterial vaginosis infections can also be mitigated by the use oflactic acid-producing (i.e., probiotic) organisms and/or theirextracellular products. As previously discussed, the cause-and-effectrelationship in bacterial vaginosis is due to the reduction of lacticacid-producing bacterial strains with the resulting multi-log increasesof to anaerobic microorganisms including, but not limited to,Gardnerella vaginalis. However, the results of a recent, 3900-womanstudy performed in Denmark demonstrated that absence of bacterialvaginosis was directly associated with sufficient vaginal colonizationof aerobic lactic acid-producing organisms. In accord, vaginalsupplementation with an effective lactic acid-producing bacterialspecies will serve to address the imbalance between aerobic lacticacid-producing organisms and the anaerobic species implicated in theetiology of bacterial vaginosis. Such vaginal supplementation may eitherbe utilized prophylactically or therapeutically.

It has now been demonstrated that certain species of lacticacid-producing bacteria can be incorporated into highly alkaline, bathproduct compositions. These compositions would prove lethal to almostall other species of lactic acid-producing bacteria including, but notlimited to: Lactobacillus, Bifidiobacterium, Enterococccus, and variousother stains of vegetative cell bacteria.

Administration remains the major problematic issue of vaginalsupplementation and, prior to the present invention, there was along-felt need for an inoculation strategy which made vaginal lacticacid supplementation incidental. The administration of an adequate doseof an effective lactic acid organisms in a bath or shower product wouldthus address some of the vaginal problems associated with frequent andeven occasional bathing, aroma-therapy, sea salt, bath powders, bathgels, bath oils and the like could contain an effective inoculation ofthe extracellular product of a lactic acid-producing bacteria for avaginal application.

The mechanics of this type of administration may be explained in thefollowing manner. After running a warm bath, the woman would add thebath product containing the therapeutic composition of the presentinvention to the water. The woman would sit in the bath, moving her legsto facilitate vaginal inoculation, for a total of approximately 20minutes. Subsequently, this treatment could be repeated on the third day(e.g., in cases of acute vuvo-vaginal candidaiasis (VVC) or bacterialvaginitis (BV)), or on a “regular basis” (i.e., at-least monthly) inorder to promote the continued stability of the vaginal ecology andmicroflora. In addition, this methodology should also prove useful inpromoting general dermal health, as some species of lacticacid-producing bacteria are useful in the promotion of healthy skin.

In another embodiment, a soft, cloth towelette soaked in a solution ofwater, potassium sorbate, disodium EDTA, Emu Oil and containing theextracellular product from a lactic acid-producing bacterial species ofthe present invention may be utilized to clean the external vaginalarea. Additional components to the aforementioned formulation mayinclude DMDM hydantoin, isopropyl myristate, methylparaben, polysorbate60, propylene glycol, propylparaben or sorbitan stearate. The disposabletowelette is used to gently wipe the perivaginal area and is thendiscarded.

In yet another embodiment, solid vaginal suppositories or insertscontaining the extracellular product from a lactic acid-producingbacterial species of the present invention and Emu Oil are utilized formucosal treatment of Candida abbicans and/or Candida tropicalisinfections. Such formulations can be made, for example, from acombination of corn starch, lactose, a metal stearate (e.g., magnesiumstearate) and povidone. Typically, one to three solid inserts should beused per day while symptoms (e.g., vaginal itch and/or whitishdischarge) are detected. Optimally, one insert per day, for a total ofthree to seven days, preferably at bedtime, is used.

In another embodiment, for an aerosol-based delivery ofmicroparticulates, an aerosol spray may be formulated by combining theextracellular product from a lactic acid-producing bacterial species ofthe present invention and Emu Oil within a carrier mixture which iscomprised of isopropyl myristate, approximately about 60% (w/w) SDalcohol 40-B, and isobutane as the propellant. A non-aerosol, manualpump spray also containing the extracellular product from a lacticacid-producing bacterial species of the present invention and Emu Oil ina neutral aqueous solution may also be utilized. A suitable sprayformulation includes alcohol, glycerin, purified water andmethylparaben, in addition to the Bacillus coagulans probioticmicroorganism.

It should also be noted that while the mitigation of yeast infections isthe primary vaginal-based utilization of Bacillus coagulans therapeuticcompositions, these compositions have also been demonstrated to behighly effective in the treatment of non-pathogenic, non-specificdermatitis. Immersion in the therapeutic bathing compositions of thepresent invention allow the establishment of the probiotic Bacilluscoagulans on the skin or mucosal membranes, which tends to mitigatedermatitis of unknown etiology.

5.6 Prevention and/or Treatment of Opportunistic Skin Infections

Opportunistic skin infections with Pseudomonas and or Staphylococcusspecies (i.e., typically Pseudomonas aeruginosa, Staphylococcusepidermidus, Staphylococcus aureus, and the like) commonly occurconcomitantly with skin allergies (e.g., allergic reactions to plantirritants such as poison ivy), bed sores, diabetic lesions or othertypes of skin lesions. Probiotic formulations containing Bacilluscoagulans spores (i.e., approximately 1×10⁵ to 1×10¹⁰/ml depending onthe specific formulation and application) and/or supernatant or filtratecontaining extracellular bacteriocins produced by Bacillus coagulans orPseudomonas lindbergii strains are highly useful in the prevention ortreatment of opportunistic skin pathogens. Additionally, probioticBacillus coagulans formulations are useful in the prevention ofinfection with Meticillin-resistant Staphylococcus aureus (MRSA),particularly following injury or invasive surgical procedures. Awater-in-oil or oil-in-water emulsion, cream, lotion, powder, aerosolpowder, or aerosol spray containing the extracellular product from alactic acid-producing bacterial species of the present invention and EmuOil is used. Various suitable carriers have been previously describedherein, and others are well-known within the art.

In the practice of this embodiment of the present invention, the skin isinitially cleaned with soap and water and dried thoroughly. Thetherapeutic composition is then applied to the skin, ensuring that thecomposition is applied to the areas between the toes, under the breasts,under the arms, or any other areas where the skin may become moist orexhibit frictional chafing or abrasion.

In addition to treating the skin topically with an emulsion, cream,lotion, powder, aerosol powder, or aerosol spray containing Bacilluscoagulans probiotic, the skin may be cleansed with a probioticformulation such as described herein.

In another embodiment of the present invention, the extracellularproduct of the selected Bacillus strain (preferably Bacillus coagulans)and/or Pseudomonas lindbergii is combined with atherapeutically-effective dose of an antibiotic and Emu Oil. Inpreferred embodiments of the present invention, the extracellularproduct of the aforementioned lactic acid-producing bacterial strains iscombined with a therapeutic concentration of one or more antibiotics,including, but not limited to: Gentamicin; Vancomycin; Oxacillin;Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin;Trimethoprim-sulfamethoxasole; a member of the Cephlosporin antibioticfamily (e.g., Cefaclor, Cefadroxil, Cefixime, Cefprozil, Ceftriaxone,Cefuroxime, Cephalexin, Loracarbef, and the like); a member of thePenicillin family of antibiotics (e.g., Ampicillin,Amoxicillin/Clavulanate, Bacampicillin, Cloxicillin, Penicillin VK, andthe like); with a member of the Fluoroquinolone family of antibiotics(e.g., Ciprofloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and the like); or amember of the Macrolide antibiotic family (e.g., Azithromycin,Erythromycin, and the like).

Equivalents

From the foregoing detailed description of the specific embodiments ofthe present invention, it should be readily apparent that unique,improved methodologies for the prevention and/or therapeutic treatmentof bacterial, fungal, yeast, and viral infections, have been disclosedherein. Although particular embodiments have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims which follow. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention as defined by the claims. Forexample, the final form (e.g., stabilized gel, cream, emulsification,and the like) which is selected for the therapeutic composition isbelieved to be a matter of routine for a person of ordinary skill in theart with knowledge of the embodiments described herein.

1. A composition comprising clotrimazole in apharmaceutically-acceptable carrier which is suitable for topicalapplication to skin or a mucous membrane of a mammal and Emu Oil.
 2. Thecomposition of claim 1, wherein said carrier is selected from a groupcomprising an emulsion, cream, lotion, gel, oil, ointment, suspension,aerosol spray, powder, aerosol powder, or semi-solid formulation.
 3. Thecomposition of claim 1, wherein the Emu Oil comprises approximately 0.5%to approximately 99.9%, by weight of said composition.
 4. Thecomposition of claim 1, wherein the Emu Oil comprises approximately 10%to approximately 75%, by weight of said composition.
 5. The compositionof claim 1, wherein the Emu Oil comprises approximately 25% toapproximately 60%, by weight of said composition.
 6. The composition ofclaim 1, further comprising one or more compounds selected from thegroup comprising dimethyl sulfoxide (DMSO) and methylsulfonylmethane(MSM).
 7. The composition of claim 1, further comprising anon-microbially-derived, anti-microbial compound selected from the groupconsisting of amphotericin B, carbol-fuchsin, ciclopirox, terbinafine,econazole, haloprogin, ketoconazole, mafenide, miconazole, naftifine,nystatin, oxiconazole, silver sulfadiazine, sulconazole, tioconazole,tolnaftate, and undecylenic acid.
 8. The composition of claim 1, furthercomprising a non-microbially-derived, anti-fungal vaginal compoundselected from the group consisting of butoconazle, econazole, gentianviolet, terbinafine, miconazole, nystatin, terconazole and tioconazole.9. The composition of claim 6, wherein said methylsulfonylmethane (MSM)is Lignisul® MSM.